RESUMO
Calreticulin (CALR) is mutated in the majority of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs). Mutant CALR (CALRdel52) exerts its effect by binding to the thrombopoietin receptor MPL to cause constitutive activation of JAK-STAT signaling. In this study, we performed an extensive mutagenesis screen of the CALR globular N-domain and revealed 2 motifs critical for CALRdel52 oncogenic activity: (1) the glycan-binding lectin motif and (2) the zinc-binding domain. Further analysis demonstrated that the zinc-binding domain was essential for formation of CALRdel52 multimers, which was a co-requisite for MPL binding. CALRdel52 variants incapable of binding zinc were unable to homomultimerize, form CALRdel52-MPL heteromeric complexes, or stimulate JAK-STAT signaling. Finally, treatment with zinc chelation disrupted CALRdel52-MPL complexes in hematopoietic cells in conjunction with preferential eradication of cells expressing CALRdel52 relative to cells expressing other MPN oncogenes. In addition, zinc chelators exhibited a therapeutic effect in preferentially impairing growth of CALRdel52-mutant erythroblasts relative to unmutated erythroblasts in primary cultures of MPN patients. Together, our data implicate zinc as an essential cofactor for CALRdel52 oncogenic activity by enabling CALRdel52 multimerization and interaction with MPL, and suggests that perturbation of intracellular zinc levels may represent a new approach to abrogate the oncogenic activity of CALRdel52 in the treatment of MPNs.
Assuntos
Calreticulina , Transtornos Mieloproliferativos , Calreticulina/genética , Humanos , Mutagênese , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Receptores de Trombopoetina/genética , ZincoRESUMO
The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of stromal antigen 2-mutant (STAG2-mutant) AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to poly(ADP-ribose) polymerase (PARP) inhibition. We developed a mouse model of MDS in which Stag2 mutations arose as clonal secondary lesions in the background of clonal hematopoiesis driven by tet methylcytosine dioxygenase 2 (Tet2) mutations and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which was associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and replication protein A complex. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Animais , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Modelos Animais de Doenças , Feminino , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Mutantes , Camundongos SCID , Camundongos Transgênicos , Síndromes Mielodisplásicas/tratamento farmacológico , Proteínas Nucleares/genética , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Células U937 , Ensaios Antitumorais Modelo de Xenoenxerto , CoesinasRESUMO
Mutations in calreticulin (CALR) are phenotypic drivers in the pathogenesis of myeloproliferative neoplasms. Mechanistic studies have demonstrated that mutant CALR binds to the thrombopoietin receptor MPL, and that the positive electrostatic charge of the mutant CALR C terminus is required for mutant CALR-mediated activation of JAK-STAT signaling. Here we demonstrate that although binding between mutant CALR and MPL is required for mutant CALR to transform hematopoietic cells; binding alone is insufficient for cytokine independent growth. We further show that the threshold of positive charge in the mutant CALR C terminus influences both binding of mutant CALR to MPL and activation of MPL signaling. We find that mutant CALR binds to the extracellular domain of MPL and that 3 tyrosine residues within the intracellular domain of MPL are required to activate signaling. With respect to mutant CALR function, we show that its lectin-dependent function is required for binding to MPL and for cytokine independent growth, whereas its chaperone and polypeptide-binding functionalities are dispensable. Together, our findings provide additional insights into the mechanism of the pathogenic mutant CALR-MPL interaction in myeloproliferative neoplasms.
